Project Summary Triple-negative breast cancer (TNBC) is a group of aggressive diseases with a greater incidence of relapse, metastasis and patient mortality compared to other breast cancer subtypes. Genetic profiling studies have identified at least six subgroups of TNBC exhibiting distinct molecular features. These subgroup- associated molecular features, or ?biomarkers?, may eventually be used to predict response to treatment; however, not all biomarkers cause tumor progression and/or treatment resistance. It is therefore critical to better understand the molecular drivers that underlie TNBC subgroups. TNBC expresses abundant Twist, a fundamental and versatile regulator of TNBC progression. Despite being a long-standing drug target, there is no approach to specifically target Twist activity. Ubiquitin is an important regulatory signal. K48-linked ubiquitination causes degradation and downregulation of protein substrates, whereas the newly characterized K63-linked ubiquitination activates protein-mediated signaling pathways. E3 ligases are the substrate recognition protein that impart specificity to the ubiquitin signal. Many E3 ligases are upregulated in human cancers. Several inhibitors that inactivate the oncogenic E3 ligases are currently being tested in clinical trials. By identifying which E3 ligases account for the K63-linked ubiquitination of Twist, we could potentially discover new drug targets that specifically block Twist to treat TNBC. The goal of this application is to characterize the K63-linked ubiquitination network that orchestrates Twist activation and protein expression and to identify the E3 ligases responsible for Twist K63-linked ubiquitination. We will conduct a series of biochemical and biological studies to uncover the mechanisms by which K63-linked ubiquitination regulates Twist activity post-translationally. We will establish the pathological roles of Twist K63-linked ubiquitination and its controlling E3 ligases in animal studies. We will validate the physiological relevance of E3 ligases as functional biomarkers in TNBC tumors and assess whether inhibiting these E3 ligases has therapeutic potential. Relevance to Public Health: Our study characterizing E3 ligases as molecular drivers of TNBC will open the door to developing new strategies for effectively treating TNBC. Through a non-biased E3 ligase screen, we identified two primary Twist-controlling E3 ligases that appear to be present in most TNBC tissues in a mutually exclusive fashion, most likely as the result of tumor heterogeneity. If the two E3 ligases, individually or in combination, do affect TNBC regulation, then they could be used as functional prognosis markers in personalized medicine. Aside from TNBC, the knowledge gained through this work is potentially applicable to other tumor types where Twist activation is implicated, such as prostrate, lung, head and neck cancers.